The expression of activated cellular oncogenes in chemically induced rat tumors and the relationship of oncogene expression to progression from the normal to the neoplastic phenotype are studied using 3T3 transfection and hybridization techniques and monoclonal antibodies directed against the specific oncogene products. Five types of tumors have been generated by single injection of F344 rats using various alkylating agents: renal mesenchymal tumors induced by methyl(methoxymethyl)-nitrosamine (DMN-OMe), intestinal adenocarcinomas induced by methyl(acetoxymethyl)-nitrosamine (DMN-OAc), hepatocellular carcinomas induced by intraportal injection of DMN-OAc followed by phenobarbital promotion, and gliomas and schwannomas induced by transplacental exposure to nitrosoethylurea (ENU). DNA purified from these tumors is utilized for 3T3 transfection assays and in Southern blot hybridizations with available oncogene probes. Tissue specific activated oncogene sequences of rat origin were found in two of these model systems. Rat renal mesenchymal tumors frequently contained K-ras (15/33); only 1/33 contained a different activated gene, and that was N-ras, a member of the same family. Schwannomas commonly contained an activated neu (erbB-2) gene (6/13); one such tumor contained both neu and N-ras. The neu gene is totally unrelated to the ras family and this finding diminishes the likelihood that selective isolation of activated oncogenes in chemically induced tumors is a technical artifact. No comparable frequency of activated genes was found in 59 chemically induced gliomas or in more than 180 hepatocellular neoplasms, which sporadically revealed N-ras (1 glioma) or K- or H-ras (six liver tumors). Oncogene proteins were localized to fixed tissue sections of tumors and non-neoplastic lesions by avidin-biotin immunocytochemistry. With monoclonal or polyclonal antibodies to the proteins or their peptides, H-ras p21 could be identified in Harvey sarcoma virus-induced sarcomas and splenic erythroblasts but not in a variety of naturally occurring and induced tumors, even those proven to have activated H-ras oncogenes.